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Endothelial cells are susceptible to rapid siRNA transfection and gene silencing ex vivo  Nicholas D. Andersen, MD, Atish Chopra, Thomas S. Monahan, MD,

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Presentation on theme: "Endothelial cells are susceptible to rapid siRNA transfection and gene silencing ex vivo  Nicholas D. Andersen, MD, Atish Chopra, Thomas S. Monahan, MD,"— Presentation transcript:

1 Endothelial cells are susceptible to rapid siRNA transfection and gene silencing ex vivo 
Nicholas D. Andersen, MD, Atish Chopra, Thomas S. Monahan, MD, Junaid Y. Malek, MD, Monica Jain, BS, Leena Pradhan, PhD, Christiane Ferran, MD, PhD, Frank W. LoGerfo, MD  Journal of Vascular Surgery  Volume 52, Issue 6, Pages (December 2010) DOI: /j.jvs Copyright © 2010 Society for Vascular Surgery Terms and Conditions

2 Fig 1 Custom pressure chamber designed to expose standard tissue culture plates to hyperbaric pressure. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

3 Fig 2 Endothelial siRNA delivery exceeds medial and adventitial delivery and is increased using distending pressure transfection. (A) Vein segments transfected with Cy5 siRNA demonstrate discrete areas of blue fluorescence independent of the red/green autofluorescence of the elastic fibers (representative micrograph, ×600). Quantitation of blue pixels by tissue layer demonstrates (B) maximal Cy5 fluorescence in the endothelium after distending pressure (DP) transfection as compared with the soak and hyperbaric pressure (HBP) transfections (*P < .05 for both comparisons). (C-E) Cy5 fluorescence in the medial layer, adventitial layer, and total vessel wall was statistically equivalent between the three transfection conditions. (F) Cumulative analysis of images from all transfection conditions demonstrates greater Cy5 fluorescence in the endothelial layer as compared to the media and the adventitia (*P < .05 for both comparisons). For all data, n = 5 to 6 vein segments per condition. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

4 Fig 3 Endothelial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein knockdown exceeds medial and adventitial GAPDH knockdown and is increased using distending pressure transfection. (A) Immunohistochemistry demonstrates GAPDH knockdown within all tissue layers after distending pressure (DP) transfection with GAPDH siRNA (panel b) as compared to vein segments treated with control siRNA (panel a). Protein knockdown is specific to GAPDH as CD31 (panels c,d) and actin (panels e,f) levels are preserved. Quantitation demonstrates (B) greater GAPDH knockdown in the endothelium using distending pressure as compared with hyperbaric (HBP) and non-pressurized (soak) transfection. (C) Medial/adventitial GAPDH levels were significantly reduced after distending pressure transfection, but not with hyperbaric or non-pressurized transfection. (D) Cumulative analysis of all transfections revealed greater knockdown in the endothelium as compared with the media/adventitia. n = 3 vein segments per condition. Micrographs (×400) correspond to one representative image of three experiments performed. (* denotes P < .05 for comparisons). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

5 Fig 4 Endothelial specific nitric oxide synthase (eNOS) knockdown is achieved using all three transfection methods and is increased using distending pressure transfection. (A) Immunohistochemistry demonstrates greatest endothelial eNOS knockdown after distending pressure (DP) transfection with eNOS siRNA (panel b) as compared to vein segments treated with control siRNA (panel a). Protein knockdown is specific to eNOS as CD31 levels (panels c,d) are preserved. (B-E) Quantitation demonstrates greater eNOS knockdown using distending pressure as compared to hyperbaric (HBP) and non-pressurized (soak) transfection. n = 3 vein segments per condition. Micrographs (×400) correspond to one representative image of three experiments performed. (* denotes P < .05 for comparisons). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

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